Epi-seq: Multiplexed ChIP-seq for personalized medicine and drug discovery

摘要 表观遗传机制的失调在肿瘤的发生和发展中起着重要作用。 几种疾病状态，包括癌症、自身免疫性、神经和炎症性疾病。 仅在癌症方面，就已鉴定出60种表观遗传调节蛋白的高频突变。喜欢 基因组测序，表观遗传信息可以在整个基因组中阐明，并可以 用于发现引起疾病或与疾病相关的表观遗传异常。的计算积分 全基因组遗传和表观遗传学图谱(表观基因分型)使新疾病的识别成为可能 亚组，儿科神经肿瘤学是一个显著的例子。开发系统解决方案， 促进表观遗传学数据集的集成是实现针对病理的个性化药物解决方案的关键 有表观遗传依赖性。染色质免疫沉淀是全基因组检测的主要方法。 DNA相关蛋白质图谱。与DNA测序的巨大进步形成鲜明对比的是 接近1,000美元基因组的技术，芯片在 工作流程，减少背景和样本量要求。此SBIR快速通道提案旨在 开发了EPI-Seq这一高吞吐量的多路复用芯片-Seq平台，以映射系统中的功能变化 表观基因组的规模和时间框架适合临床和药物筛选应用。具有自动化功能 考虑到兼容性，Epi-Seq结合了使用PIXUL的固定染色质的样品制备和增溶， 一台96孔平板超声仪，可在不到30分钟内剪切96个染色质样品，带有转座酶- 辅助多重芯片-序列(-芯片)高通量地对整个基因组进行功能注释 格式化。通过一组重组AbFlex®芯片合格抗体靶向实现多路传输 表观遗传景观的各种标志性特征，每一种特征都与转座酶结合- 通过定点连接的相容条形码寡核苷酸。第一阶段可行性将使用PIXUL从 人和小鼠组织中AIM1和芯片激活的AbFlex®抗体与关键DNA结合的比较 传统方法和芯片序列方法中的蛋白质和组蛋白PTM。第二阶段的产品开发工作将 优化-目标3和目标4中的芯片目标多路复用将努力使当前的工作流程实现自动化 兼容性，从而实现了免提的“样品到序列”工作流程。第二阶段的工作将结束 由独立实验室进行阿尔法测试，其反馈将整合到后续 Active Motif的商业化努力。EPI-Seq是一种有潜力加速的使能技术 对各种人类疾病的表观遗传学诊断、预后和治疗的发展，并将是 作为合同服务和产品提供，独立销售或与PIXUL仪器捆绑销售。

SUMMARY Dysregulation of epigenetic mechanisms is known to play important roles in the development and progression several disease states including cancer, autoimmune, neurological and inflammatory disorders. In cancer alone, high frequency mutations in sixty epigenetic regulatory proteins have been identified. Like genome sequencing, epigenetic information can be elucidated across the entire genome and profiling can be used to find epigenetic abnormalities that cause or correlate with disease. Computational integration of genome-wide genetic and epigenetic profiles (epi-genotyping) has enabled identification of novel disease subgroups, with pediatric neuro-oncology being a notable example. Developing a systems solution that facilitates integration of epigenetic data sets is key to enabling personalized medicine solutions for pathologies with epigenetic dependencies. Chromatin immunoprecipitation (ChIP) is the primary method for genome-wide mapping of DNA associated proteins. In contrast to the tremendous advances in DNA sequencing technologies, which are approaching the $1,000 genome, ChIP has only had incremental improvements in work flow, reduction of background and sample size requirements. This SBIR Fast Track proposal intends to develop Epi-Seq, a high throughput multiplexing ChIP-Seq platform to map functional alterations in the epigenome on a scale and time frame suitable for clinical and drug screening applications. With automation compatibility in mind, Epi-Seq combines sample preparation and solubilization of fixed chromatin using PIXUL, a 96-well plate sonicator capable of shearing 96 chromatin samples in less than 30 minutes, with Transposase- Assisted Multiplex ChIP-Seq (TAM-ChIP) to functionally annotate the entire genome in a high throughput format. Multiplexing is achieved with a panel of recombinant AbFlex® ChIP-qualified antibodies targeting various hallmark features of the epigenetic landscape, each of which has been conjugated to a transposase- compatible barcoded oligonucleotide via site-directed conjugation. Phase I feasibility will use PIXUL from human and mouse tissues in Aim1 and compare TAM-ChIP enabled AbFlex® antibodies for key DNA binding proteins and histone PTMs in traditional and TAM-ChIP-seq methods. Phase II product development efforts will optimize TAM-ChIP target multiplexing in Aim 3 and Aim 4 efforts will adapt current workflows for automation compatibility such that a hands-free “sample-to-sequence” workflow is achieved. Phase II efforts will conclude with alpha-testing by independent laboratories whose feedback will be integrated into subsequent commercialization efforts by Active Motif. Epi-Seq is an enabling technology with potential accelerate development of epigenetic diagnostics, prognostic and therapeutics for diverse human pathologies and will be offered both as a contract service and as a product, sold independently or bundled with the PIXUL instrument.